The long range goal of this proposed research is to characterize in molecular terms the mechanism of threonine inhibition of the aspartokinase-homoserine dehydrogenase-I enzyme complex. Special emphasis will be placed upon delineating the mechanisms which produce the complex interactions between the two enzymatic activities of this two-headed enzyme. The transition of the enzyme from active to inhibited forms caused by threonine binding will be characterized by studying the energetics of this process as well as by seeking to determine whether dimensional alterations occur within the native tetramer. A key long term goal of this research is to attempt to define critical changes in the active site of the enzyme which account for the allosteric inhibition of the enzyme by threonine. The proposed studies are directed towards establishing a valid structural model for the enzyme which will provide a basis for understanding the complex allosteric effects observed. The electron microscopic and optical diffraction studies proposed will form the framework for such a model, hopefully providing critical information regarding subunit packing, symmetry properties and basic overall dimensions of the tetramer. Such data should be extremely helpful in the interpretation of the proposed experiments utilizing paramagnetic and fluorescent probes. Use of a combination of paramagnetic and fluorescent probes can provide direct information regarding the proximity of the two active sites and the nature of their interaction. The thermodynamic studies proposed will provide the basis for distinguishing between various co-operative models of the allosteric changes in the enzyme. The eventual correlation of data obtained from the approaches outlined below should give greater insight into the nature of the allosteric transition in the AK-HSDH-I molecule. BIBLIOGRAPHIC REFERENCES: K. Wright and M. Takahashi. "The Threonine Sensitive Aspartokinase of E. coli: Affinity Labelling & Effector Binding to a Modified Enzyme." Abstracts Tenth Int. Congress. of Biochemistry, Hamburg, Germany, July, 1976. Abstract of talk to be presented at the 172nd National Meeting of the ACS. August, 1976. K. Wright and M. Takahashi. "Affinity Labeling of Aspartokinase Via Formation of Inert Co (III) Substrate & Inhibitor Adducts."